Protein toxins active against lepidopteran pests

ABSTRACT

Disclosed and claimed are novel Bacillus thuringiensis isolates which have lepidopteran activity. Thus, these isolates, or mutants thereof, can be used to control such insect pests. Further, genes encoding novel δ-endotoxins can be removed from the isolates and transferred to other host microbes, or plants. Expression of the δ-endotoxins in such hosts results in the control of susceptible insect pests in the environment of such hosts.

CROSS-REFERENCE TO A RELATED APPLICATION

This is a continuation of application Ser. No. 08/291,368, filed Aug.15, 1994 now U.S. Pat. No. 5,686,069 which is a continuation-in-part ofapplication Ser. No. 08/032,778, filed Mar 16, 1993 now abandoned, whichis a continuation of application Ser. No. 07/597,607, filed Oct. 15,1990, now abandoned.

BACKGROUND OF THE INVENTION

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive,spore-forming bacterium characterized by parasporal crystalline proteininclusions. These inclusions often appear microscopically asdistinctively shaped crystals. These crystalline proteins can beproforms of δ-endotoxins which are highly toxic to pests and specific intheir toxic activity. Certain B.t. endotoxin genes have been isolatedand sequenced, and recombinant DNA-based B.t. products have beenproduced and approved. In addition, with the use of genetic engineeringtechniques, new approaches for delivering B.t. endotoxins toagricultural environments are under development, including the use ofplants genetically engineered with endotoxin genes for insect resistanceand the use of stabilized intact microbial cells as B.t. endotoxindelivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4-S7).Thus, isolated B.t. endotoxin genes are becoming commercially valuable.

Until the last ten years, commercial use of B.t. pesticides has beenlargely restricted to a narrow range of lepidopteran (caterpillar)pests. Preparations of the spores and crystals of B.thuringiensis subsp.kurstaki have been used for many years as commercial insecticides forlepidopteran pests. For example, B.thuringiensis var. kurstaki HD-1produces a delta endotoxin which is toxic to the larvae of a number oflepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticideswith specificities for a much broader range of pests. For example,subspecies of B.t., namely israelensis and san diego (a.k.a. B.t.tenebrionis, a.k.a. M-7), have been used commercially to control insectsof the orders Diptera and Coleoptera, respectively (Gaertner, F. H.[1989] "Cellular Delivery Systems for Insecticidal Proteins: Living andNon-Living Microorganisms," in Controlled Delivery of Crop ProtectionAgents, R. M. Wilkins, ed., Taylor and Francis, New York and London,1990, pp. 245-255). See also Couch, T. L. (1980) "Mosquito Pathogenicityof Bacillus thuringiensis var. israelensis," Developments in IndustrialMicrobiology 22:61-76; Beegle, C. C., (1978) "Use of EntomogenousBacteria in Agroecosystems," Developments in Industrial Microbiology20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter(1983) Z. ang. Ent 96:500-508, describe a B.t. isolate named Bacillusthuringiensis var. tenebrionis, which is reportedly active against twobeetles in the order Coleoptera. These are the Colorado potato beetle,Leptinotarsa decemlineata, and Agelastica alni.

Recently, new subspecies of B.t. have been identified, and genesresponsible for active δ-endotoxin proteins have been isolated (Hofte,H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hofteand Whiteley classified B.t. crystal protein genes into 4 major classes.The classes were CryI (Lepidoptera-specific),CryII (Lepidoptera- andDiptera-specific), CryIII (Coleoptera-specific), and CryIV(Diptera-specific). The discovery of strains specifically toxic to otherpests has been reported. (Feitelson, J. S., J. Payne, L. Kim [1992]Bio/Technology 10:271-275).

The cloning and expression of a B.t. crystal protein gene in Escherichiacoli has been described in the published literature (Schnepf, H. E., H.R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat.No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expressionof B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,797,276 and4,853,331 disclose B.thuringiensis var. san diego (a.k.a. B.t.tenebrionis, a.k.a. M-7) which can be used to control coleopteranpestsin various environments. U.S. Pat. No. 5,164,180 discloses a B.t.isolate, PS81A2, which is active against lepidopteran pests. U.S. Pat.No. 5,151,363 discloses certain isolates of B.t. which have activityagainst nematodes. Many other patents have issued for new B.t. isolatesand new uses of B.t. isolates. The discovery of new B.t. isolates andnew uses of known B.t. isolates remains an empirical, unpredictable art.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns novel Bacillus thuringiensis isolateswhich have activity against lepidopteran pests.

Specifically, the invention comprises novel B.t. isolates and mutantsthereof, and novel delta endotoxin genes obtainable from these B.t.isolates which encode proteins which are active against lepidopteranpests.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the one-letter amino acid sequence of the Generic Formula(SEQ ID NO. 27). Numbering is for convenience and approximate locationonly. In the Generic Formula, the N-terminal half of the molecule iscomprised of residue nos. 1-638. The terminal half is comprised ofresidues 639-1213. Wherein

    ______________________________________                                        A = ala     G = gly    M = met    S = ser                                     C = cys     H = his    N = asn    T = thr                                     D = asp     I = ile    P = pro    V = val                                     E = glu     K = lys    Q = gln    W = trp                                     F = phe     L = leu    R = arg    Y = tyr                                     k = K or R                                                                    z = G, S, D, or N                                                             j = E, Q, R, or K                                                             x = G, S, D, N, E, Q, R, or K                                                 u = C, P, T, or A                                                             b = M, I, L, V, or F                                                          o = C, P, T, A, M, I, L, V, or F                                              ______________________________________                                         - = any naturally occurring amino acid                                        . = any naturally occurring amino acid or complete omission thereof.     

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is the nucleotide sequence of the gene 81A2.

SEQ ID NO. 2 is the amino acid sequence of the toxin 81A2.

SEQ ID NO. 3 is the nucleotide sequence of the gene 91C2.

SEQ ID NO. 4 is the amino acid sequence of the toxin 91C2.

SEQ ID NO. 5 is a radiolabeled oligonucleotide probe used in RFLPanalysis as described in Example 3.

SEQ ID NO. 6 is a forward oligonucleotide primer used to amplify gene91C2 according to the subject invention.

SEQ ID NO. 7 is a reverse oligonucleotide primer used to amplify gene91C2 according to the subject invention.

SEQ ID NO. 8 is a synthetic oligonucleotide probe used to identify gene91C2 according to the subject invention.

SEQ ID NO. 9 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 10 is a nucleotide probe according to the subject invention.

SEQ ID NO. 11 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 12 is a nucleotide probe according to the subject invention.

SEQ ID NO. 13 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 14 is a nucleotide probe according to the subject invention.

SEQ ID NO. 15 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 16 is a nucleotide probe according to the subject invention.

SEQ ID NO. 17 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 18 is a nucleotide probe according to the subject invention.

SEQ ID NO. 19 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 20 is a nucleotide probe according to the subject invention.

SEQ ID NO. 21 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 22 is a nucleotide probe according to the subject invention.

SEQ ID NO. 23 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 24 is a nucleotide probe according to the subject invention.

SEQ ID NO. 25 is the peptide sequence encoded by probes for CryIF genes.

SEQ ID NO. 26 is a nucleotide probe according to the subject invention.

SEQ ID NO. 27 is the Generic Formula according to the subject invention.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns isolates of Bacillus thuringiensis havinganti-lepidopteran activity. These isolates comprise genes which code forδ-endotoxins, which toxins are responsible for the observedanti-lepidopteran activity. Thus, the subject invention concernsanti-lepidopteran B.t. isolates, anti-lepidopteran B.t. toxins, andgenes which encode these toxins. Further embodiments of the subjectinvention concern recombinant hosts transformed with genes encoding theanti-lepidopteran B.t. toxins.. The subject invention further concernsmethods for controlling lepidopterans, said methods comprising the useof the isolates, toxins, genes, and recombinant hosts of the subjectinvention.

Specifically exemplified herein are the isolates designated B.t. PS81T1,B.t. PS53C2, B.t. PS31F4, B.t. PS86V1, B.t. PS 8612, B.t. PS73E, B.t.PS81K, B.t. PS83E2, B.t. PS81E, B.t. PS81Z3, B.t. PS53B5, B.t. PS83R,B.t. PS53B2, Bt. PS83N2, B.t. PS81B5, B.t. PS86W1, and B.t. PS93C2. Alsospecifically exemplified is the toxin designated 91C2 and the gene whichencodes this toxin. The 91C2 gene is a CryIF gene. CryIF is a subclassof genes within the lepidopteran-active CryI class of B.t. genes. Thediscovery described in the subject application enables a person skilledin the art to identify other CryIF toxins (and genes coding for thesetoxins) having anti-lepidopteran activity. The toxins of the subjectinvention are characterized as being active against lepidopterans andhaving one or more of the following characteristics:

1. A high degree of amino acid homology with toxin 91C2.

2. A nucleotide sequence encoding the toxin wherein the nucleotidesequence hybridizes with probes or genes disclosed herein.

3. A nucleotide sequence encoding the toxin wherein the nucleotidesequence can be amplified by PCR using primers disclosed herein.

4. An amino acid sequence which conforms to the Generic Formulapresented herein.

5. Immunoreactivity to an antibody raised to toxin 91 C2.

Bacillus thuringiensis isolates useful according to the subjectinvention have the following characteristics in their biologically pureform:

                  TABLE 1                                                         ______________________________________                                        Taxonomic characterization of the B.t. isolates of the subject invention      Approx. Toxin                                                                 Isolate                                                                              Crystal Type                                                                             MW (kD)   Serotype Activity                                 ______________________________________                                        PS81T1 bipyramid  130       aizawai  Lepidoptera                              PS53C2 bipyramid  130, 60   kurstaki Lepidoptera                              PS31F4 bipyramid  130, 60   kurstaki Lepidoptera                              PS86V1 bipyramid  130       galleriae                                                                              Lepidoptera                              PS86I2 bipyramid  130       morrisoni                                                                              Lepidoptera                              PS73E  bipyramid  130       aizawai  Lepidoptera                              PS81K  bipyramid  130       aizawai  Lepidoptera                              PS83E2 amorphic   130       aizawai  Lepidoptera                              PS81E  bipyramid  130       aizawai  Lepidoptera                              PS81Z3 bipyramid  130       aizawai  Lepidoptera                              PS53B5 bipyramid  130, 60   kenyae   Lepidoptera                              PS83R  bipyramid  130       aizawai  Lepidoptera                              PS53B2 bipyramid  130, 60   kenyae   Lepidoptera                              PS83N2 bipyramid  130, 60   sotto/kenyae                                                                           Lepidoptera                              PS81B5 amorphic   130       aizawai  Lepidoptera                              PS86W1 bipyramid  130       galleriae                                                                              Lepidoptera                              PS91C2 bipyramid  130       morrisoni                                                                              Lepidoptera                              ______________________________________                                    

B.t. isolates useful according to the subject invention have beendeposited. Also deposited are recombinant microbes comprising the B.t.genes of interest. The cultures have been deposited in the permanentcollection of the Patent Culture Collection (NRRL), Regional ResearchCenter, 1815 North University Street, Peoria, Ill. 61604 USA.

    ______________________________________                                        Culture        Accession Number                                                                           Deposit Date                                      ______________________________________                                        Bacillus thuringiensis PS81IA                                                                NRRL B-18484 April 19, 1989                                    Bacillus thuringiensis PS91C2                                                                NRRL B-18931 December 27, 1991                                 E. Coli NM522 (pMYC2361)                                                                     NRRL B-21016N                                                                              December 17, 1992                                 ______________________________________                                    

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14and 35 U.S.C.§122. The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of the deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposits should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Toxins and genes. The toxins and genes according to the subjectinvention include not only the full length sequences disclosed hereinbut also fragments of these sequences, longer sequences, and fusionproteins, which retain the characteristic pesticidal activity of thetoxins specifically exemplified herein.

One aspect of the subject invention concerns the discovery of a genericchemical formula hereinafter referred to as the Generic Formula (SEQ IDNO.27), which can be used to identify toxins having activity againstlepidopterans. The Generic Formula describes toxin proteins havingmolecular weights of about 130 kDa.

The Generic Formula is shown in FIG. 1 designated by a one-letter aminoacid sequence. The Sequence Listing provided herein according to thePatentIn format utilizes the three-letter amino acid code and has noprovision for showing a choice between two amino acids at a givenposition. Therefore, within the PatentIn Sequence Listing, "Xaa" is usedto denote points of variation within a sequence, but the single lettercode of FIG. 1 should be referred to for the specific amino acidsubstitutions which are acceptable at a given location in the sequence.

Further guidance for characterizing the lepidopteran toxins of thesubject invention is provided in Tables 2 and 3, which demonstrate therelatedness among toxins within the known CryI subclasses oflepidopteran toxins. These tables show a numeric score for the bestmatching alignment between two proteins that reflects: (1) positivescores for exact matches, (2) positive or negative scores reflecting thelikelihood (or not) of one amino acid substituting for another in arelated protein, and (3) negative scores for the introduction of gaps. Aprotein sequence aligned to itself will have the highest possible-score,i.e., all exact matches and no gaps. However, an unrelated protein or arandomly generated sequence will typically have a low positive score.Related sequences have scores between the random background score andthe perfect match score.

The sequence comparisons reported herein were made using the algorithmof Smith and Waterman ([1981] Advances in Applied Mathematics2:482-489), implemented as the program "Bestfit" in the GCG SequenceAnalysis Software Package Version 7, April 1991. The sequences werecompared with default parameter values (comparison: Swagappep.Cmp, Gap:3.0, length weight: 0.1). The program output value is referred to as theQuality score.

Tables 2 and 3 show the pairwise alignment scores between the indicatedamino acids of the CryI toxin proteins. Table 4 shows the amino acidscompared from the proteins of interest.

Table 2 shows the scores prior to adjustment for random sequence scores.Note that for each subclass, the highest alignment score is always withanother toxin protein from the same subclass. For example, the highestalignment score with CryIA(a), aside from itself, is with CryIA(d).Furthermore, CryIA(a) scores highest with all three other CryIA toxinproteins. In a similar manner, other CryI toxins score highest withother members of the same subclass. Of particular relevance to thesubject invention is the fact that the CryIF toxin proteins scorehighest with each other.

Table 3 shows the same analysis after subtraction of the average scoreof 50 alignments of random shuffles of the column sequences with the rowsequences. Note that in Table 3 the same relationships hold as in Table2, i.e., toxin proteins score highest with other members of the samesubclass. Again, the two CryIF toxin proteins score highest with eachother. Examination of the adjusted alignment scores for members of thesame subclass reveals that CryI subclasses can be defined as thoseproteins with adjusted alignment scores of about 450 or greater.

Thus, certain toxins of the subject invention can be defined as thosewhich have lepidopteran activity and have an alignment value of 450-500or greater with CryIF(a) or CryIF(b) (91C2). As used herein, the term"alignment value" refers to the adjusted scores obtained above and usedto create the scores reported in Table 3.

                                      TABLE 2                                     __________________________________________________________________________    Raw quality scores                                                                    CryIA(a)                                                                           CryIA(b)                                                                           CryIA(c)                                                                           CryIA(d)                                                                           CryIB                                                                             CryIC                                                                             CryID                                                                             CryIE(a)                                                                           CryIE(b)                                                                           CryIF(a)                                                                           CryIF(b)               __________________________________________________________________________                                                           (91C2)                 CryIA(a)                                                                              911  819  706  857  426 519 533 536  535  532  557                    CryIA(b)     912  785  790  428 512 540 547  546  543  565                    CryIA(c)          914  679  390 482 508 512  533  502  501                    CryIA(d)               911  422 514 539 549  538  539  559                    CryIB                       954 428 410 408  375  421  434                    CryIC                           926 525 547  480  494  495                    CryID                               888 505  499  507  497                    CryIE(a)                                902  722  494  487                    CryIE(b)                                     899  480  477                    CryIF(a)                                          902  803                    CryIF(b) (91C2)                                        900                    __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Net quality scores                                                                    CryIA(a)                                                                           CryIA(b)                                                                           CryIA(c)                                                                           CryIA(d)                                                                           CryIB                                                                             CryIC                                                                             CryID                                                                             CryIE(a)                                                                           CryIE(b)                                                                           CryIF(a)                                                                           CryIF(b)               __________________________________________________________________________                                                           (91C2)                 CryIA(a)                                                                              724  633  520  671  240 332 352 351  350  349  373                    CryIA(b)     726  600  606  241 327 359 362  360  360  383                    CryIA(c)          728  493  204 295 327 328  347  319  317                    CryIA(d)               727  236 328 357 363  353  356  377                    CryIB                       763 240 229 223  189  235  249                    CryIC                           738 343 361  293  309  309                    CryID                               710 325  319  328  318                    CryIE(a)                                717  538  310  304                    CryIE(b)                                     713  296  294                    CryIF(a)                                          719  620                    CryIF(b) (91C2)                                        713                    __________________________________________________________________________

                  TABLE 4                                                         ______________________________________                                        Protein       Amino acids compared                                            ______________________________________                                        CryIA(a)      1-607                                                           CryIA(b)      1-608                                                           CryIA(c)      1-609                                                           CryIA(d)      1-607                                                           CryIB         1-636                                                           CryIC         1-617                                                           CryID         1-592                                                           CryIE(a)      1-601                                                           CryIE(b)      1-599                                                           CryIF(a)      1-601                                                           CryIF(b) (91C2)                                                                             1-600                                                           ______________________________________                                    

Toxins of the subject invention are specifically exemplified herein bythe toxin encoded by the gene designated 91C2. Since this toxin ismerely exemplary of the toxins of the subject invention, it should bereadily apparent that the subject invention further comprises varianttoxins (and nucleotide sequences coding for variant toxins) having thesame, or essentially the same, biological activity against lepidopteransof 91C2. These equivalent toxins will have amino acid homology with91C2. This amino acid homology will typically be greater than 75%,preferably be greater than 90%, and most preferably be greater than 95%.The amino acid homology will be highest in certain critical regions ofthe toxin which account for biological activity or are involved in thedetermination of three-dimensional configuration which ultimately isresponsible for the biological activity. In this regard, certain aminoacid substitutions are acceptable and can be readily made in regionswhich are not critical to activity or are conservative amino acidsubstitutions which do not affect the three-dimensional configuration ofthe molecule. For example, amino acids may be placed in the followingclasses: non-polar, uncharged polar, basic, and acidic. Conservativesubstitutions whereby an amino acid of one class is replaced withanother amino acid of the same type fall within the scope of the subjectinvention so long as the substitution does not materially alter thebiological activity of the compound. Table 5 provides a listing ofexamples of amino acids belonging to each class.

                  TABLE 5                                                         ______________________________________                                        Class of Amino Acid                                                                          Examples of Amino Acids                                        ______________________________________                                        Nonpolar       Ala, Val, Leu, Ile, Pro, Met, Phe, Trp                         Uncharged Polar                                                                              Gly, Ser, Thr, Cys, Tyr, Asn, Gln                              Acidic         Asp, Glu                                                       Basic          Lys, Arg, His                                                  ______________________________________                                    

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

The toxins of the subject invention can also be characterized in termsof the shape and location of toxin inclusions, which are describedabove.

It should be apparent to a person skilled in this art that genesencoding lepidopteran-active toxins can be identified and obtainedthrough several means. The specific genes exemplified herein may beobtained from the isolates deposited at a culture depository asdescribed above. These genes, or portions or variants thereof, may alsobe constructed synthetically, for example, by use of a gene machine. Asused herein, the terms "variants" or "variations" of genes refer tonucleotide sequences which code for the same toxins or which code forequivalent toxins having lepidopteran activity. Variations of thesegenes may be readily constructed using standard techniques for makingpoint mutations. Also, fragments of these genes can be made usingcommercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Also, genes which code for active fragments may beobtained using a variety of other restriction enzymes. Proteases may beused to directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can alsobe located from B.t. isolates and/or DNA libraries using the teachingsprovided herein. There are a number of methods for obtaining thepesticidal toxins of the instant invention. For example, antibodies tothe pesticidal toxins disclosed and claimed herein can be used toidentify and isolate other toxins from a mixture of proteins.Specifically, antibodies may be raised to the portions of the toxinswhich are most constant and most distinct from other B.t. toxins. Theseantibodies can then be used to specifically identify equivalent toxinswith the characteristic activity by immunoprecipitation, enzyme linkedimmunosorbent assay (ELISA), or Western blotting. Antibodies to thetoxins disclosed herein, or to equivalent toxins, or fragments of thesetoxins, can readily be prepared using standard procedures in this art.The genes coding for these toxins can then be obtained from themicroorganism.

A further method for identifying the toxins and genes of the subjectinvention is through the use of oligonucleotide probes. These probes aredetectable nucleotide sequences. These sequences may be detectable byvirtue of an appropriate label or may be made inherently fluorescent asdescribed in International Patent Application No. WO93/16094. As is wellknown in the art, if the probe molecule and nucleic acid samplehybridize by forming a strong bond between the two molecules, it can bereasonably assumed that the probe and sample have substantial homology.Detection of the probe provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying toxin-encoding genes of thesubject invention.

The nucleotide segments which are used as probes according to theinvention can be synthesized by use of DNA synthesizers using standardprocedures. In the use of labeled nucleotide segments as probes, theparticular probe is labeled with any suitable label known to thoseskilled in the art, including radioactive and non-radioactive labels.Typical radioactive labels include ³² P, ¹²⁵ I, ³⁵ S, or the like. Aprobe labeled with a radioactive isotope can be constructed from anucleotide sequence complementary to the DNA sample by a conventionalnick translation reaction, using a DNase and DNA polymerase. The probeand sample can then be combined in a hybridization buffer solution andheld at an appropriate temperature until annealing occurs. Preferably,hybridization is conducted under stringent conditions by techniques wellknown in the art, as described, for example, in Keller, G. H., M. M.Manak (1989) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.Thereafter, the membrane is washed free of extraneous materials, leavingthe sample and bound probe molecules typically detected and quantifiedby autoradiography and/or liquid scintillation counting.

Non-radioactive labels include, for example, ligands such as biotin orthyroxine, as well as enzymes such as hydrolases or perixodases, or thevarious chemiluminescers such as luciferin, or fluorescent compoundslike fluorescein and its derivatives. The probe may also be labeled atboth ends with different types of labels for ease of separation, as, forexample, by using an isotopic label at the end mentioned above and abiotin label at the other end.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid; a certain degree of mismatch can betolerated. Therefore, the probes of the subject invention includemutations (both single and multiple), deletions, insertions of thedescribed sequences, and combinations thereof wherein said mutations,insertions and deletions permit formation of stable hybrids with thetarget polynucleotide of interest. Mutations, insertions, and deletionscan be produced in a given polynucleotide sequence in many ways, andthese methods are known to an ordinarily skilled artisan. Other methodsmay become known in the future.

The known methods include, but are not limited to:

(1) synthesizing chemically or otherwise an artificial sequence which isa mutation, insertion or deletion of the known sequence;

(2) using a probe of the present invention to obtain via hybridization anew sequence or a mutation, insertion or deletion of the probe sequence;and

(3) mutating, inserting or deleting a test sequence in vitro or in vivo.

It is important to note that the mutational, insertional, and deletionalvariants generated from a given probe may be more or less efficient thanthe original probe. Notwithstanding such differences in efficiency,these variants are within the scope of the present invention.

Thus, mutational, insertional, and deletional variants of the disclosedsequences can be readily prepared by methods which are well known tothose skilled in the art. These variants can be used in the same manneras the instant probes so long as the variants have substantial sequencehomology with the probes. As used herein, substantial sequence homologyrefers to homology which is sufficient to enable the variant to functionin the same capacity as the original probe. Preferably, this homology isgreater than 50%; more preferably, this homology is greater than 75%;and most preferably, this homology is greater than 90%. The degree ofhomology needed for the variant to function in its intended capacitywill depend upon the intended use of the sequence. It is well within theskill of a person trained in this art to make mutational, insertional,and deletional mutations which are designed to improve the function ofthe sequence or otherwise provide a methodological advantage.

Specific nucleotide probes useful according to the subject invention inthe rapid identification of CryIF class toxin genes include:

(i) DNA coding for a peptide sequence "Ser Thr Gly Arg Leu Pro Leu Asp"(SEQ ID NO. 9). A specific example of such a probe is "AGTACWGGMAGRTTACCRTT RGAY" (SEQ ID NO. 10);

(ii) DNA coding for a peptide sequence "Glu Asp Ser Pro Val Ser Ala Asn"(SEQ ID NO. 11). A specific example of such a probe is "GARGATTCWCCAGTWTCWGC WAAT" (SEQ ID NO. 12);

(iii) DNA coding for a peptide sequence "Asn Gly Phe Asn Arg Ala Glu PheGly Val" (SEQ ID NO. 13). A specific example of such a probe is"AATGGWTTTA ATAGTGCTGAATTTGGGAGTW" (SEQ ID NO. 14);

(iv) DNA coding for a peptide sequence "Val Thr Ala Glu Thr Val Arg SerGln Thr" (SEQ ID NO. 15). A specific example of such a probe is"GTAACWGCAG ARACWGTWAG WAGTCAAACW" (SEQ ID NO. 16);

(v) DNA coding for a peptide sequence "Val Phe Asn Pro Gly Gly Ala IleTrp Ile Ala Asp Glu" (SEQ ID NO. 17). A specific example of such a probeis "GTMTTYAATC CWGGWGGMGCMATWTGGATWGCWGATGARG AT" (SEQ ID NO. 18);

(vi) DNA coding for a peptide sequence "Val Arg Gly Gly Phe Gly" (SEQ IDNO. 19). A specific example of such a probe is "GTMMGAGGWG GWTTTGGR"(SEQ ID NO. 20);

(vii) DNA coding for a peptide sequence "Gly Thr Asn His Thr Arg Thr"(SEQ ID NO. 21). A specific example of such a probe is "GGWACRAAYCAYACMMGAAC W" (SEQ ID NO. 22);

(viii) DNA coding for a peptide sequence "Val Arg Trp Pro Gly Glu Ile"(SEQ ID NO. 23). A specific example of such a probe is "GTWMGATGGCCWGGWGARAT W" (SEQ ID NO. 24);

(ix) DNA coding for a peptide sequence "Ser Asp Ser Trp Arg Ala" (SEQ IDNO. 25). A specific example of such a probe is "AGTGATTCWT GGAGAGCW"(SEQ ID NO. 26).

Because of the redundancy of the genetic code, i.e., more than onecoding nucleotide triplet (codon) can be used for most of the aminoacids used to make proteins, different nucleotide sequences can code fora particular amino acid. Thus, the amino acid sequences of the B.t.toxins and peptides can be prepared by equivalent nucleotide sequencesencoding the same amino acid sequence of the protein or peptide.Accordingly, the subject invention includes such equivalent nucleotidesequences. Also, inverse or complement sequences are an aspect of thesubject invention and can be readily used by a person skilled in thisart.

Recombinant hosts. The toxin-encoding genes harbored by the isolates ofthe subject invention can be introduced into a wide variety of microbialor plant hosts. Expression of the toxin gene results, directly orindirectly, in the intracellular production and maintenance of thepesticide. With suitable microbial hosts, e.g., Pseudomonas, livemicrobes can be applied to the situs of lepidopterans where they willproliferate and be ingested by the pest. The result is a control of thispest. Alternatively, the microbe hosting the toxin gene can be treatedunder conditions that prolong the activity of the toxin and stabilizethe cell. The treated cell, which retains the toxic activity, then canbe applied to the environment of the target pest.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. For example,microorganism hosts can be selected which are known to occupy the soil.These microorganisms are selected so as to be capable of successfullycompeting in the soil with the wild-type microorganisms. It is alsoimportant that they provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, provide forimproved protection of the pesticide from environmental degradation andinactivation.

A large number of microorganisms are known to inhabit the rhizosphere(the soil surrounding plant roots). These microorganisms includebacteria, algae, and fungi. Of particular interest are microorganisms,such as bacteria, e.g., genera Bacillus, Pseudomonas, Erwinia, Serratia,Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter,Azotobacter, Leuconostoc, Alcaligenes and Clostridium; fungi,particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium;microalgae, e.g., families Cyanophyceae, Prochlorophyceae, Rhodophyceae,Dinophyceae, Chrysophyceae, Prymnesiophyceae, Xanthophyceae,Raphidophyceae, Bacillariophyceae, Eustigmatophyceae, Cryptophyceae,Euglenophyceae, Prasinophyceae, and Chlorophyceae. Of particularinterest are such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum,Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonascampestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobactervinlandii; and phytosphere yeast species such as Rhodotorula rubra, R.glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens,C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae,Sporobolomycesroseus, S. odorus, Kluyveromycesveronae, and Aureobasidiumpollulans. Of particular interest are the pigmented microorganisms.

A wide variety of ways are available for introducing a B.t. geneencoding a toxin into a microorganism host under conditions which allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Treatment of cells. As mentioned above, B.t. or recombinant cellsexpressing a B.t. toxin can be treated to prolong the toxin activity andstabilize the cell. The pesticide microcapsule that is formed comprisesthe B.t. toxin within a cellular structure that has been stabilized andwill protect the toxin when the microcapsule is applied to theenvironment of the target pest. Suitable host cells may include eitherprokaryotes or eukaryotes, normally being limited to those cells whichdo not produce substances toxic to higher organisms, such as mammals.However, organisms which produce substances toxic to higher organismscould be used, where the toxic substances are unstable or the level ofapplication sufficiently low as to avoid any possibility of toxicity toa mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t.toxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability of protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as formaldehyde andglutaraldehyde; anti-infectives, such as zephiran chloride andcetylpyridinium chloride; alcohols, such as isopropyl and ethanol;various histologic fixatives, such as Lugol iodine, Bouin's fixative,and Helly's fixative (See: Humason, Gretchen L., Animal TissueTechniques, W. H. Freeman and Company, 1967); or a combination ofphysical (heat) and chemical agents that preserve and prolong theactivity of the toxin produced in the cell when the cell is administeredto the host's environment. In one preferred embodiment, acids can beused to stabilize the cells. Examples of physical means are shortwavelength radiation such as gamma-radiation and X-radiation, freezing,UV irradiation, lyophilization, and the like. Methods for treatment ofmicrobial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462,which are incorporated herein by reference.

The cells generally will have enhanced structural stability which willenhance resistance to environmental conditions. Where the pesticide isin a proform, the method of cell treatment should be selected so as notto inhibit processing of the proform to the mature form of the pesticideby the target pest pathogen. For example, formaldehyde will crosslinkproteins and could inhibit processing of the proform of a polypeptidepesticide. The method of treatment should retain a substantial portionof the bio-availability or bioactivity of the toxin.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; survival in aqueous environments; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Growth of cells. The cellular host containing the B.t. insecticidal genemay be grown in any convenient nutrient medium, where the DNA constructprovides a selective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells of the invention can be cultured using standard art mediaand fermentation techniques. Upon completion of the fermentation cyclethe bacteria can be harvested by first separating the B.t. spores andcrystals from the fermentation broth by means well known in the art. Therecovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers, and othercomponents to facilitate handling and application for particular targetpests. These formulations and application procedures are all well knownin the art.

Formulations. Formulated bait granules containing an attractant andspores and crystals of the B.t. isolates, or recombinant microbescomprising the gene(s) obtainable from the B.t. isolates disclosedherein, can be applied to the soil. Formulated product can also beapplied as a seed-coating or root treatment or total plant treatment atlater stages of the crop cycle.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1-95% by weight of the pesticide while the liquid formulations willgenerally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the lepidopteran,e.g., soil, by spraying, dusting, sprinkling, or the like.

Mutants. Mutants of the novel isolates of the invention can be made byprocedures well known in the art. For example, an asporogenous mutantcan be obtained through ethylmethane sulfonate (EMS) mutagenesis of anovel isolate. The mutants can be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

A smaller percentage of the asporogenous mutants will remain intact andnot lyse for extended fermentation periods; these strains are designatedlysis minus (-). Lysis minus strains can be identified by screeningasporogenous mutants in shake flask media and selecting those mutantsthat are still intact and contain toxin crystals at the end of thefermentation. Lysis minus strains are suitable for a cell fixationprocess that will yield a protected, encapsulated toxin protein.

To prepare a phage resistant variant of said asporogenous mutant, analiquot of the phage lysate is spread onto nutrient agar and allowed todry. An aliquot of the phage sensitive bacterial strain is then plateddirectly over the dried lysate and allowed to dry. The plates areincubated at 30° C. The plates are incubated for 2 days and, at thattime, numerous colonies could be seen growing on the agar. Some of thesecolonies are picked and subcultured onto nutrient agar plates. Theseapparent resistant cultures are tested for resistance by cross streakingwith the phage lysate. A line of the phage lysate is streaked on theplate and allowed to dry. The presumptive resistant cultures are thenstreaked across the phage line. Resistant bacterial cultures show nolysis anywhere in the streak across the phage line after overnightincubation at 30° C. The resistance to phage is then reconfirmed byplating a lawn of the resistant culture onto a nutrient agar plate. Thesensitive strain is also plated in the same manner to serve as thepositive control. After drying, a drop of the phage lysate is plated inthe center of the plate and allowed to dry. Resistant cultures showed nolysis in the area where the phage lysate has been placed afterincubation at 30° C. for 24 hours.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing of the B.t. Isolates of the Invention

A subculture of a novel B.t. isolate, or mutants thereof, can be used toinoculate the following medium, a peptone, glucose, salts medium.

    ______________________________________                                        Bacto Peptone        7.5    g/l                                               Glucose              1.0    g/l                                               KH.sub.2 PO.sub.4    3.4    g/l                                               K.sub.2 HPO.sub.4    4.35   g/l                                               Salt Solution        5.0    ml/l                                              CaCl.sub.2 Solution  5.0    ml/l                                              ______________________________________                                    

Salts Solution (100 ml)

    ______________________________________                                               MgSO.sub.4 ·7H.sub.2 O                                                        2.46 g                                                               MnSO.sub.4 ·H.sub.2 O                                                         0.04 g                                                               ZnSO.sub.4 ·7H.sub.2 O                                                        0.28 g                                                               FeSO.sub.4 ·7H.sub.2 O                                                        0.40 g                                                        ______________________________________                                    

CaCl₂ Solution (100 ml)

    ______________________________________                                               CaCl.sub.2 ·2H.sub.2 O                                                       3.66 g                                                                pH 7.2                                                                 ______________________________________                                    

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30 ° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and/or crystals, obtained in the above fermentation, canbe isolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Activity of B.t. Isolates Against Lepidopterans

The following strains have been tested for anti-lepidopteran activitywith the following results:

                  TABLE 6                                                         ______________________________________                                        Bioassay results                                                                        % Mortality                                                         Strain      Trichoplusia ni                                                                            Spodoptera exigua                                    ______________________________________                                        PS81T1      96,8                                                              PS53C2      100, 100                                                          PS31F4                   100, 100                                             PS86V1      100                                                               PS86I2      100, 92                                                           PS73E       100, 100                                                          PS81K       100, 100                                                          PS83E2      100, 100                                                          PS81E       100, 92                                                           PS81Z3      100                                                               PS53B5                   100                                                  PS83R       100                                                               PS53B2                   100                                                  PS83N2      100                                                               PS81B5      100, 100                                                          PS86W1      100                                                               PS91C2      100, 100                                                          PS81A2      100, 100                                                          ______________________________________                                    

Spodoptera exigua bioassay procedure. B.t. cultures were harvested andresuspended in sterile deionized water. Fixed volumes of each culturewere incorporated into USDA Insect Diet (Technical Bulletin 1528, U.S.Department of Agriculture, 1976). Twenty-four neonate S. exigua wereexposed to the diet for 6 days. Mortality readings were taken at thistime.

Trichoplusia ni bioassay procedure. B.t. cultures were harvested andresuspended in sterile deionized water. Fixed volumes of each culturewere top loaded onto USDA Insect Diet. Trays were infested with neonateT. ni. After 6 days mortality was determined.

EXAMPLE 3 Characterization of Toxin Genes by RFLP Analysis

Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cellsgrown to an optical density, at 600 mn, of 1.0. The cells were recoveredby centrifugation, and protoplasts were prepared in TES buffer (30 mMTris-HCl, 10 mM EDTA, 50 mM NaCl, pH=8.0) containing 20% sucrose and 50mg/ml lysozyme. The protoplasts were lysed by addition of SDS to a finalconcentration of 4%. The cellular material was precipitated overnight at4° C. in 100 mM (final concentration) neutral potassium chloride. Thesupernate was extracted twice with phenol/chloroform(1:1). The DNA wasprecipitated with ethanol and purified by isopycnic banding on a cesiumchloride-ethidium bromide gradient.

Total cellular DNA isolated from B.t. cells was digested with arestriction endonuclease and separated by electrophoresison a 0.8% (w/v)agarose-TAE (50 mM Tris-HCl, 20 mM NaOAc, 2.5 mM EDTA, pH=8.0) bufferedgel. A Southern blot of the gel was hybridized with the [³²P]-radiolabeled oligonucleotide probe, ATGATTCATGCGGCAGATA (SEQ ID NO.5), and then washed to remove unbound radioactivity. The blot wasexposed to KODAK X-OMAT™ film using standard autoradiography techniques.The results are an array of hybridizing bands (fingerprint) whichcorrespond to toxin genes or toxin gene fragments. This type ofcharacterization is known as Restriction Fragment Length Polymorphism(RFLP) analysis which classifies each isolate by a distinct DNAfingerprint.

                  TABLE 7                                                         ______________________________________                                        DNA fingerprints for B.t. isolates of the subject invention                   Isolate   Hybridizing HindIII Fragments (Kb)                                  ______________________________________                                        PS81T1    1.13, 3.0, 9.4                                                      PS53C2    1.052, 5.8, 6.6                                                     PS31F4    5.5, 8.0                                                            PS86V1    5.5, 6.0, 6.6                                                       PS86I2    5.0, 6.6, 7.5, 12                                                   PS73E     1.052, 1.13, 3.0, 8.5                                               PS81K     3.2, 7.5, 9.4, 13                                                   PS83E2    3.2, 8.5, 12                                                        PS81E     1.13, 3.2, 9.4                                                      PS81Z3    1.13, 3.0, 8.5                                                      PS53B5    1.13, 3.0, 7.5                                                      PS83R     1.13, 3.0, 8.5, 12                                                  PS53B2    1.052, 1.13, 3.0, 7.5                                               PS83N2    5.5                                                                 PS81B5    8.0, 13                                                             PS86W1    5.5, 6.6                                                            PS91C2    1.13, 3.0, 6.0, 7.5, 8.5                                            PS81A2    13, 16                                                              ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Hybridizing HindIII fragments of B.t. isolates                                of the subject invention                                                      Isolate  Novel Hybridizing HindIII Fragments (˜Kb)                      ______________________________________                                        PS91C2   3.0, 6.0, 7.5                                                        PS83E2   3.2                                                                  PS86I2   5.0                                                                  PS31F4   5.5, 8.0                                                             PS53C2   5.8                                                                  PS81T1   9.4                                                                  ______________________________________                                    

EXAMPLE 4 Molecular Cloning and Expression of a Novel CryIF Toxin Genefrom Bacillus thuringiensis Strain PS91C2

Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cellsgrown to an optical density, at 600 nm, of 1.0. Cells were pelleted bycentrifugation and resuspended in protoplast buffer (20 mg/ml lysozymein 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After incubationat 37° C. for 1 hour, protoplasts were lysed by two cycles of freezingand thawing. Nine volumes of a solution of 0.1 M NaCl, 0.1% SDS, 0.1 MTris-Cl were added to complete lysis. The cleared lysate was extractedtwice with phenol:chloroform (1:1). Nucleic acids were precipitated withtwo volumes of ethanol and pelleted by centrifugation. The pellet wasresuspended in TE buffer and RNase was added to a final concentration of50 μg/ml. After incubation at 37° C. for 1 hour, the solution wasextracted once each with phenol:chloroform (1:1) and TE-saturatedchloroform. DNA was precipitated from the aqueous phase by the additionof one-tenth volume of 3 M NaOAc and two volumes of ethanol. DNA waspelleted by centrifugation, washed with 70% ethanol, dried, andresuspended in TE buffer.

A 1.58 kbp fragment of the novel 130 kDa toxin gene was obtained bypolymerase chain reaction (PCR) amplificationfrom PS91C2 cellular DNAusing the following primers: forward5'-GAGTGGGAAGCAGATCTTAATAATGCACAATTAAGG-3' (SEQ ID NO. 6) and reverse5'-ATAC(C or T)CGATCGATATGATA(G or A)TCCGT-3' (SEQ ID NO. 7). This DNAfragment was cloned into pBluescript S/K (Stratagene, La Jolla, Calif.)and the DNA sequence determined by dideoxynucleotide sequencingmethodology (Sanger et al. [1977] Proc. Natl. Acad. Sci USA74:5463-5467) using Sequenase (U.S. Biochemicals, Cleveland, Ohio). DNAsequences unique to the CryIF gene were identified by computercomparison with other CryI genes. An oligonucleotide probe with thefollowing sequence was synthesized: 5'-CCCAATGTGAATGTACTTTGCGC-3' (SEQID NO. 8). This probe was radiolabeled with ³² P and used in standardhybridizations of Southern blots of PS91 C2 total cellular DNA.Hybridizing bands included an approximately 7.5 kbp HindIII fragment.

A gene library was constructed from PS91C2 DNA partially digested withNdeII. Partial restriction digests were fractionated by agarose gelelectrophoresis. DNA fragments 9.3 to 23 kbp in size were excised fromthe gel, electroeluted from the gel slice, purified on an Elutip D ionexchange column (Schleicher and Schuell, Keene, N.H.), and recovered byethanol precipitation. The NdeII inserts were ligated intoBamHI-digested LambdaGem-11 (Promega, Madison, Wis.). Recombinant phagewere packaged and plated on E. coli KW251 cells. Plaques were screenedby hybridization with each of the respective probes described above.Hybridizing phage were plaque-purified and used to infect liquidcultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

For subcloning the gene encoding the 130 kDa CryIF toxin, preparativeamounts of phage DNA were digested with Sau3A and electrophoresed onagarose gel. The approximately 8 kbp band containing the toxin gene wasexcised from the gel, electroeluted from the gel slice, and purified byion exchange chromatography as described above. The purified DNA insertwas ligated into an XhoI-digested pHTBlueII (an E. coli/B. thuringiensisshuttle vector comprised of pBluescript S/K (Stratagene) and thereplication origin from a resident B.t. plasmid (D. Lereclus et al.[1989] FEMS Microbiol. Lett. 60:211-218). The ligation mix was used totransform frozen, competent E. coli NM522 cells (ATCC 47000).β-galactosidase transformants were screened by restriction digestion ofalkaline lysate plasmid minipreps as above. The desired plasmidconstruct, pMYC2361, contains a toxin gene that is novel compared toother toxin genes containing insecticidal proteins.

pMYC2361 was introduced into the acrystalliferous (Cry⁻) B.t. host, CryB(A. Aronson, Purdue University, West Lafayette, Ind.) byelectroporation. Expression of the 130 kDa toxin was demonstrated bySDS-PAGE analysis. NaBr-purified crystals were prepared (Pfannenstiel,M. A. et al. [1984] FEMS Microbiol. Lett. 21:39) for determination oftoxicity of the cloned gene product to Plutella xylostella by thescreening method described in Example 3. The LC₅₀ for the CryIF toxinagainst P. xylostella was determined to be 5 μg toxin/ml diet.

EXAMPLE 5 Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants withgenes encoding a lepidopteran toxin. The transformed plants areresistant to attack by lepidopterans.

Genes encoding lepidopteran-active toxins, as disclosed herein, can beinserted into plant cells using a variety of techniques which are wellknown in the art. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher plants. The vectors comprise, forexample, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly,the sequence encoding the B.t. toxin can be inserted into the vector ata suitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 0 120 516;Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerijKanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. PlantSci. 4:1-46; and An et al. (1985) EMBO J. 4:277-287.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, or electroporation as well asother possible methods. If agrobacteria are used for the transformation,the DNA to be inserted has to be cloned into special plasmids, namelyeither into an intermediate vector or into a binary vector. Theintermediate vectors can be integrated into the Ti or Ri plasmid byhomologous recombination owing to sequences that are homologous tosequences in the T-DNA. The Ti or Ri plasmid also comprises the virregion necessary for the transfer of the T-DNA. Intermediate vectorscannot replicate themselves in agrobacteria. The intermediate vector canbe transferred into Agrobacterium tumefaciens by means of a helperplasmid (conjugation). Binary vectors can replicate themselves both inE. coli and in agrobacteria. They comprise a selection marker gene and alinker or polylinker which are framed by the right and left T-DNA borderregions. They can be transformed directly into agrobacteria (Holstersetal. [1978] Mol. Gen. Genet. 163:181-187). The agrobacterium used as hostcell is to comprise a plasmid carrying a vir region. The vir region isnecessary for the transfer of the T-DNA into the plant cell. AdditionalT-DNA may be contained. The bacterium so transformed is used for thetransformation of plant cells. Plant explants can advantageously becultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenesfor the transfer of the DNA into the plant cell. Whole plants can thenbe regenerated from the infected plant material (for example, pieces ofleaf, segments of stalk, roots, but also protoplasts orsuspension-cultivated cells) in a suitable medium, which may containantibiotics or biocides for selection. The plants so obtained can thenbe tested for the presence of the inserted DNA. No special demands aremade of the plasmids in the case of injection and electroporation. It ispossible to use ordinary plasmids, such as, for example, pUCderivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

EXAMPLE 6 Cloning of Novel B.t. Genes Into Insect Viruses

A number of viruses are known to infect insects. These viruses include,for example, baculoviruses and entomopoxviruses. In one embodiment ofthe subject invention, lepidopteran-active genes, as described herein,can be placed with the genome of the insect virus, thus enhancing thepathogenicity of the virus. Methods for constructing insect viruseswhich comprise B.t. toxin genes are well known and readily practiced bythose skilled in the art. These procedures are described, for example,in Merryweather et al. (Merryweather, A. T., U. Weyer, M. P. G. Harris,M. Hirst, T. Booth, R. D. Possee [1990] J. Gen. Virol. 71:1535-1544) andMartens et al. (Martens, J. W. M., G. Honee, D. Zuidema, J. W. M. vanLent, B. Visser, J. M. Vlak [1990] Appl. Environmental Microbiol.56(9):2764-2770).

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 27                                            - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3522 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #thuringiensisORGANISM: Bacillus                                                        (B) STRAIN: aizawai                                                 #PS81A2   (C) INDIVIDUAL ISOLATE:                                             -    (vii) IMMEDIATE SOURCE:                                                  #- 11 (tm) Library of August Sick                                                       (B) CLONE: 81A2                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - ATGGAGAATA ATATTGAAAA TCAATGCATA CCTTACAATT GTTTAAATAA TC - #CTGAAGTA         60                                                                          - GAGATATTAG GGATTGAAAG GTCAAATAGT AACGTAGCAG CAGAAATCGG CT - #TGGGGCTT        120                                                                          - AGTCGTCTGC TCGTTTCCCG AATTCCACTA GGGGATTTTA TACTTGGCTT GT - #TTGATGTA        180                                                                          - ATATGGGGGG CTATAGGTCC TTCACAATGG GATATATTTT TAGAGCAAAT TG - #AGCTATTG        240                                                                          - ATCGGCCAAA GAATAGAGGA ATTCGCTAGG AATCAGGCAA TTTCTAGATT AC - #AAGGGCTA        300                                                                          - AGCAATCTTT ACCGAATTTA CACAAATGCT TTTAAAAACT GGGAAGTAGA TC - #CTACTAAT        360                                                                          - CCAGCATTAA GAGAAGAGAT GCGTATTCAA TTTAATGACA TGAACAGTGC TC - #TTACAACA        420                                                                          - GCTATTCCTC TTTTTTCAGT TCAAGGTTAT GAAATTCCTC TTTTATCAGT AT - #ATGTTCAA        480                                                                          - GCTGCAAATT TACATTTATC GGTTTTGAGA GATGTTTCAG TGTTTGGACA AC - #GTTGGGGA        540                                                                          - TTTGATGTAG CAACAATCAA TAGTCGTTAT AATGATTTAA CTAGGCTTAT TG - #GCGAATAT        600                                                                          - ACTGATTATG CTGTACGTTG GTATAATACG GGGTTAAATC GTTTACCACG TA - #ATGAAGGG        660                                                                          - GTACGAGGAT GGGCAAGATT TAATAGGTTT AGAAGAGAGT TAACAATATC AG - #TATTAGAT        720                                                                          - ATTATTTCTT TTTTCCAAAA TTACGATTCT AGATTATATC CAATTCCGAC AA - #TCTATCAA        780                                                                          - TTAACGCGGG AAGTATATAC AGATCCGGTA ATTAATATAA CTGATTATAG AG - #TTACCCCA        840                                                                          - AGTTTCGAGA GTATTGAAAA TTCAGCTATT AGAAGTCCCC ATCTTATGGA TT - #TCTTAAAT        900                                                                          - AATATAATTA TTGACACTGA TTTAATTAGA GGCGTTCACT ATTGGGCGGG GC - #ATCGTGTA        960                                                                          - ACTTCTCATT TTACCGGTAG TTCGCAAGTG ATAAGCTCCC CTCAATACGG GA - #TAACTGCA       1020                                                                          - AACGCAGAAC CGAGTCGAAC TATTGCTCCT AGCACTTTTC CAGGTCTTAA TC - #TATTTTAT       1080                                                                          - AGAACACTAT CAGACCCTTT CTTCCGAAGA TCCGATAATA TTATGCCAAC AT - #TAGGAATA       1140                                                                          - AATGTAGTGC AGGGGGTAGG ATTCATTCAA CCAAATAATG GTGAAGTTCT AT - #ATAGAAGG       1200                                                                          - AGAGGAACAG TAGATTCTCT TGATGAGTTG CCAATTGACG GTGAGAATTC AT - #TAGTTGGA       1260                                                                          - TATAGTCATA GATTAAGTCA CGTTACATTA ACCAGGTCGT TATATAATAC TA - #ATATAACT       1320                                                                          - AGCTTGCCAA CATTTGTTTG GACACATCAC AGTGCTACTG ATCGAAATAT AA - #TCTATCCG       1380                                                                          - GATGTAATTA CACAAATACC ATTGGTAAAA TCATTCTCCC TTACTTCAGG TA - #CCTCTGTA       1440                                                                          - GTCAGAGGCC CAGGATTTAC AGGAGGGGAT ATCATCCGAA CTAACGTTAA TG - #GTAATGTA       1500                                                                          - CTAAGTATGA GTCTTAATTT TAGTAATACA TCATTACAGC GGTATCGCGT GA - #GAGTTCGT       1560                                                                          - TATGCTGCTT CTCAAACAAT GGTCATGAGA GTAAATGTTG GAGGGAGTAC TA - #CTTTTGAT       1620                                                                          - CAAGGATTCC CTAGTACTAT GAGTGCAAAT GGGTCTTTGA CATCTCAATC AT - #TTAGATTT       1680                                                                          - GCAGAATTTC CTGTAGGCAT TAGTACATCT GGCAGTCAAA CTGCTGGAAT AA - #GTATAAGT       1740                                                                          - AATAATCCAG GTAGACAAAC GTTTCACTTA GATAGAATTG AATTTATCCC AG - #TTGATGCA       1800                                                                          - ACATTTGAAG CAGAATATGA TTTAGAAAGA GCACAAAAGG CGGTGAATTC GC - #TGTTTACT       1860                                                                          - TCTTCCAATC AAATCGAGTT AAAAACAGAT GTGACGGATT ATCATATTGA TC - #AAGTATCC       1920                                                                          - AATTTAGTAG ATTGTTTATC CGATGAATTT TGTCTGGATG AAAAGCGAGA AT - #TGTCCGAG       1980                                                                          - AAAGTCAAAC ATGCGAAGCG ACTCAGTGAT GAGCGGAATT TACTTCAAGA TC - #CAAACTTC       2040                                                                          - AGAGGGATCA ATAGGCAACC AGACCGTGGC TGGAGAGGAA GTACGGATAT TA - #CCATCCAA       2100                                                                          - GGAGGAGATG ACGTATTCAA AGAGAATTAC GTCACACTAC CAGGTACCTT TG - #ATGAGTGC       2160                                                                          - TATCCAACGT ATTTGTATCA AAAAATAGAT GAGTCGAAAT TAAAAGCCTA TA - #ACCGTTAC       2220                                                                          - CAATTAAGAG GGTATATCGA AGATAGTCAA GACTTAGAAA TCTATTTAAT TC - #GCTACAAT       2280                                                                          - GCAAAACACG AAACAGTAAA TGTACCAGGT ACGGGTTCCT TATGGCCGCT TT - #CAGTCGAA       2340                                                                          - AGTCCAATTG GAAGGTGTGG AGAACCGAAT CGGTGTGTGC CACACCTTGA AT - #GGAATCCT       2400                                                                          - GATTTAGATT GTTCCTGCAG AGACGGGGAA AAATGTGCAC ATCATTCCCA TC - #ATTTCTCC       2460                                                                          - TTGGACATTG ATGTTGGATG CACAGACTTG CAAGAGGATC TAGGCGTGTG GG - #TTGTATTC       2520                                                                          - AAGATTAAGA CGCAGGAAGG TTATGCAAGA TTAGGAAATC TGGAATTTAT CG - #AAGAGAAA       2580                                                                          - CCATTAATTG GAGAAGCACT GTCTCGTGTG AAGAGAGCGG AAAAAAAATG GA - #GAGACAAA       2640                                                                          - CGGGAAAAAC TACAATTGGA AACAAAACGA GTATATACAG AGGCAAAAGA AG - #CTGTGGAT       2700                                                                          - GCTTTATTCG TAGATTCTCA ATATGATAGA TTACAAGCAG ATACAAACAT TG - #GTATGATT       2760                                                                          - CATGCGGCAG ATAGACTTGT TCATCAGATC CACGAGGCTT ATCTTCCAGA AC - #TACCTTTC       2820                                                                          - ATTCCAGGAA TAAATGTGGT GATTTTTGAA GAATTAGAAA ACCGTATTTC TA - #CTGCATTA       2880                                                                          - TCCCTATATG ATGCGAGAAA TGTCATTAAA AATGGCGATT TCAATAATGG CT - #TATCATGC       2940                                                                          - TGGAACGTGA AAGGGCATGT AGATGTAGTA GAACAAAACA ACCACCGTTC GG - #TCCTTGTT       3000                                                                          - GTCCCGGAAT GGGAAGCAGA AGTGTCACAA ACAATTCGTG TCTGTCCGGG GC - #GTGGCTAT       3060                                                                          - ATCCTCCGTG TTACAGCGTA CAAAGAGGGA TATGGAGAAG GTTGCGTAAC CA - #TCCATGAG       3120                                                                          - ATCGAGAACA ATACAGACGA ACTAAAATTT AAAAACTGTG AAGAAGAGGA AG - #TGTATCCA       3180                                                                          - ACGGATACAG GAACGTGTAA TGATTATACT GCACACCAAG GTACAGCAGG AT - #CCACAGAT       3240                                                                          - TCATGTAATT CCCGTAATAT CAGATATGAG GATGCATATG AAATGAATAC TA - #CAGCATCT       3300                                                                          - GTTAATTACA AACCGACTTA CGAAGAAGAA AGGTATACAG ATGTACAAGG AG - #ATAATCAT       3360                                                                          - TGTGAATATG ACAGAGGGTA TGTGAATTAT CGACCAGTAC CAGCTGGTTA TG - #TGACAAAA       3420                                                                          - GAATTAGAGT ACTTCCCAGA AACCGATAAG GTATGGATTG AGATCGGAGA AA - #CGGAAGGG       3480                                                                          #3522              ATGT CGAATTACTC CTTATGGAGG AA                              - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1174 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #thuringiensisORGANISM: Bacillus                                                        (B) STRAIN: aizawai                                                 #PS81A2   (C) INDIVIDUAL ISOLATE:                                             -    (vii) IMMEDIATE SOURCE:                                                  #- 11 (tm) Library of August Sick                                                       (B) CLONE: 81A2                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Glu Asn Asn Ile Glu Asn Gln Cys Ile Pr - #o Tyr Asn Cys Leu Asn         #                15                                                           - Asn Pro Glu Val Glu Ile Leu Gly Ile Glu Ar - #g Ser Asn Ser Asn Val         #            30                                                               - Ala Ala Glu Ile Gly Leu Gly Leu Ser Arg Le - #u Leu Val Ser Arg Ile         #        45                                                                   - Pro Leu Gly Asp Phe Ile Leu Gly Leu Phe As - #p Val Ile Trp Gly Ala         #    60                                                                       - Ile Gly Pro Ser Gln Trp Asp Ile Phe Leu Gl - #u Gln Ile Glu Leu Leu         #80                                                                           - Ile Gly Gln Arg Ile Glu Glu Phe Ala Arg As - #n Gln Ala Ile Ser Arg         #                95                                                           - Leu Gln Gly Leu Ser Asn Leu Tyr Arg Ile Ty - #r Thr Asn Ala Phe Lys         #           110                                                               - Asn Trp Glu Val Asp Pro Thr Asn Pro Ala Le - #u Arg Glu Glu Met Arg         #       125                                                                   - Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Th - #r Thr Ala Ile Pro Leu         #   140                                                                       - Phe Ser Val Gln Gly Tyr Glu Ile Pro Leu Le - #u Ser Val Tyr Val Gln         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Ala Ala Asn Leu His Leu Ser Val Leu Arg As - #p Val Ser Val Phe Gly         #               175                                                           - Gln Arg Trp Gly Phe Asp Val Ala Thr Ile As - #n Ser Arg Tyr Asn Asp         #           190                                                               - Leu Thr Arg Leu Ile Gly Glu Tyr Thr Asp Ty - #r Ala Val Arg Trp Tyr         #       205                                                                   - Asn Thr Gly Leu Asn Arg Leu Pro Arg Asn Gl - #u Gly Val Arg Gly Trp         #   220                                                                       - Ala Arg Phe Asn Arg Phe Arg Arg Glu Leu Th - #r Ile Ser Val Leu Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Ile Ser Phe Phe Gln Asn Tyr Asp Ser Ar - #g Leu Tyr Pro Ile Pro         #               255                                                           - Thr Ile Tyr Gln Leu Thr Arg Glu Val Tyr Th - #r Asp Pro Val Ile Asn         #           270                                                               - Ile Thr Asp Tyr Arg Val Thr Pro Ser Phe Gl - #u Ser Ile Glu Asn Ser         #       285                                                                   - Ala Ile Arg Ser Pro His Leu Met Asp Phe Le - #u Asn Asn Ile Ile Ile         #   300                                                                       - Asp Thr Asp Leu Ile Arg Gly Val His Tyr Tr - #p Ala Gly His Arg Val         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Thr Ser His Phe Thr Gly Ser Ser Gln Val Il - #e Ser Ser Pro Gln Tyr         #               335                                                           - Gly Ile Thr Ala Asn Ala Glu Pro Ser Arg Th - #r Ile Ala Pro Ser Thr         #           350                                                               - Phe Pro Gly Leu Asn Leu Phe Tyr Arg Thr Le - #u Ser Asp Pro Phe Phe         #       365                                                                   - Arg Arg Ser Asp Asn Ile Met Pro Thr Leu Gl - #y Ile Asn Val Val Gln         #   380                                                                       - Gly Val Gly Phe Ile Gln Pro Asn Asn Gly Gl - #u Val Leu Tyr Arg Arg         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Arg Gly Thr Val Asp Ser Leu Asp Glu Leu Pr - #o Ile Asp Gly Glu Asn         #               415                                                           - Ser Leu Val Gly Tyr Ser His Arg Leu Ser Hi - #s Val Thr Leu Thr Arg         #           430                                                               - Ser Leu Tyr Asn Thr Asn Ile Thr Ser Leu Pr - #o Thr Phe Val Trp Thr         #       445                                                                   - His His Ser Ala Thr Asp Arg Asn Ile Ile Ty - #r Pro Asp Val Ile Thr         #   460                                                                       - Gln Ile Pro Leu Val Lys Ser Phe Ser Leu Th - #r Ser Gly Thr Ser Val         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Arg Gly Pro Gly Phe Thr Gly Gly Asp Il - #e Ile Arg Thr Asn Val         #               495                                                           - Asn Gly Asn Val Leu Ser Met Ser Leu Asn Ph - #e Ser Asn Thr Ser Leu         #           510                                                               - Gln Arg Tyr Arg Val Arg Val Arg Tyr Ala Al - #a Ser Gln Thr Met Val         #       525                                                                   - Met Arg Val Asn Val Gly Gly Ser Thr Thr Ph - #e Asp Gln Gly Phe Pro         #   540                                                                       - Ser Thr Met Ser Ala Asn Gly Ser Leu Thr Se - #r Gln Ser Phe Arg Phe         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Ala Glu Phe Pro Val Gly Ile Ser Thr Ser Gl - #y Ser Gln Thr Ala Gly         #               575                                                           - Ile Ser Ile Ser Asn Asn Pro Gly Arg Gln Th - #r Phe His Leu Asp Arg         #           590                                                               - Ile Glu Phe Ile Pro Val Asp Ala Thr Phe Gl - #u Ala Glu Tyr Asp Leu         #       605                                                                   - Glu Arg Ala Gln Lys Ala Val Asn Ser Leu Ph - #e Thr Ser Ser Asn Gln         #   620                                                                       - Ile Glu Leu Lys Thr Asp Val Thr Asp Tyr Hi - #s Ile Asp Gln Val Ser         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Asn Leu Val Asp Cys Leu Ser Asp Glu Phe Cy - #s Leu Asp Glu Lys Arg         #               655                                                           - Glu Leu Ser Glu Lys Val Lys His Ala Lys Ar - #g Leu Ser Asp Glu Arg         #           670                                                               - Asn Leu Leu Gln Asp Pro Asn Phe Arg Gly Il - #e Asn Arg Gln Pro Asp         #       685                                                                   - Arg Gly Trp Arg Gly Ser Thr Asp Ile Thr Il - #e Gln Gly Gly Asp Asp         #   700                                                                       - Val Phe Lys Glu Asn Tyr Val Thr Leu Pro Gl - #y Thr Phe Asp Glu Cys         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Gl - #u Ser Lys Leu Lys Ala         #               735                                                           - Tyr Asn Arg Tyr Gln Leu Arg Gly Tyr Ile Gl - #u Asp Ser Gln Asp Leu         #           750                                                               - Glu Ile Tyr Leu Ile Arg Tyr Asn Ala Lys Hi - #s Glu Thr Val Asn Val         #       765                                                                   - Pro Gly Thr Gly Ser Leu Trp Pro Leu Ser Va - #l Glu Ser Pro Ile Gly         #   780                                                                       - Arg Cys Gly Glu Pro Asn Arg Cys Val Pro Hi - #s Leu Glu Trp Asn Pro         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asp Leu Asp Cys Ser Cys Arg Asp Gly Glu Ly - #s Cys Ala His His Ser         #               815                                                           - His His Phe Ser Leu Asp Ile Asp Val Gly Cy - #s Thr Asp Leu Gln Glu         #           830                                                               - Asp Leu Gly Val Trp Val Val Phe Lys Ile Ly - #s Thr Gln Glu Gly Tyr         #       845                                                                   - Ala Arg Leu Gly Asn Leu Glu Phe Ile Glu Gl - #u Lys Pro Leu Ile Gly         #   860                                                                       - Glu Ala Leu Ser Arg Val Lys Arg Ala Glu Ly - #s Lys Trp Arg Asp Lys         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Arg Glu Lys Leu Gln Leu Glu Thr Lys Arg Va - #l Tyr Thr Glu Ala Lys         #               895                                                           - Glu Ala Val Asp Ala Leu Phe Val Asp Ser Gl - #n Tyr Asp Arg Leu Gln         #           910                                                               - Ala Asp Thr Asn Ile Gly Met Ile His Ala Al - #a Asp Arg Leu Val His         #       925                                                                   - Gln Ile His Glu Ala Tyr Leu Pro Glu Leu Pr - #o Phe Ile Pro Gly Ile         #   940                                                                       - Asn Val Val Ile Phe Glu Glu Leu Glu Asn Ar - #g Ile Ser Thr Ala Leu         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Ser Leu Tyr Asp Ala Arg Asn Val Ile Lys As - #n Gly Asp Phe Asn Asn         #               975                                                           - Gly Leu Ser Cys Trp Asn Val Lys Gly His Va - #l Asp Val Val Glu Gln         #           990                                                               - Asn Asn His Arg Ser Val Leu Val Val Pro Gl - #u Trp Glu Ala Glu Val         #      10050                                                                  - Ser Gln Thr Ile Arg Val Cys Pro Gly Arg Gl - #y Tyr Ile Leu Arg Val         #  10205                                                                      - Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gly Cy - #s Val Thr Ile His Glu         #               10401030 - #                1035                              - Ile Glu Asn Asn Thr Asp Glu Leu Lys Phe Ly - #s Asn Cys Glu Glu Glu         #              10550                                                          - Glu Val Tyr Pro Thr Asp Thr Gly Thr Cys As - #n Asp Tyr Thr Ala His         #          10705                                                              - Gln Gly Thr Ala Gly Ser Thr Asp Ser Cys As - #n Ser Arg Asn Ile Arg         #      10850                                                                  - Tyr Glu Asp Ala Tyr Glu Met Asn Thr Thr Al - #a Ser Val Asn Tyr Lys         #  11005                                                                      - Pro Thr Tyr Glu Glu Glu Arg Tyr Thr Asp Va - #l Gln Gly Asp Asn His         #               11201110 - #                1115                              - Cys Glu Tyr Asp Arg Gly Tyr Val Asn Tyr Ar - #g Pro Val Pro Ala Gly         #              11350                                                          - Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Gl - #u Thr Asp Lys Val Trp         #          11505                                                              - Ile Glu Ile Gly Glu Thr Glu Gly Lys Phe Il - #e Val Asp Asn Val Glu         #      11650                                                                  - Leu Leu Leu Met Glu Glu                                                         1170                                                                      - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 3504 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (genomic)                                       -    (iii) HYPOTHETICAL: NO                                                   -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #thuringiensisORGANISM: Bacillus                                                        (B) STRAIN: Morrissoni                                              #PS91C2   (C) INDIVIDUAL ISOLATE:                                             -    (vii) IMMEDIATE SOURCE:                                                   11 Library of Teresa: LambdaGem                                                             Thompson                                                                 (B) CLONE: 91C2                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - ATGAAGAATA ACATTCAAAA TCAATGCGTA CCTTACAATT GTTTAAGTAA TC - #CTGAAGTA         60                                                                          - GAAATATTAA GTGAAGAAAG AAGTACTGGC AGATTACCGT TAGATATATC CT - #TGTCGCTT        120                                                                          - ACACGTTTCC TTTTGAGTGA ATTTGTTCCA GGTGTGGGAG TTGCGTTTGG AT - #TATTTGAT        180                                                                          - TTAATATGGG GTTTTATAAC TCCTTCTGAA TGGAGTTTAT TTCTTTTACA GA - #TTGAACAA        240                                                                          - CTGATTGAAC AAAGAATTGA AACATTGGAA AGGAACCGGG CAATTACTAC AT - #TACGAGGG        300                                                                          - TTAGCGGATA GCTATGAAGT TTACCTTGAG GCACTAAGAG AGTGGGAAGA AA - #ATCCTAAT        360                                                                          - AATGCACAAT TAAGGGAAGA TGTGCGTATT CGATTTGCTA ATACAGACGA CG - #CTTTAATA        420                                                                          - ACAGCAATAA ATAATTTTAC ACTTACAAGT TTTGAAATCC CTCTTTTATC GG - #TCTATGTT        480                                                                          - CAAGCGGCGA ATCTACATTT ATCACTATTA AGAGATGCTG TATCGTTTGG GC - #AGGGTTGG        540                                                                          - GGGCTGGATA TAGCTACTGT TAATAATCAT TATAATAGAT TAATAAATCT TA - #TTCATAGA        600                                                                          - TATACGGAAC ATTGTTTGGA CACATACAAT CAAGGATTAG AAAACTTAAG AG - #GTACTAAT        660                                                                          - ACTCGACAAT GGTCAAGATT CAATCAGTTT AGGAGAGAGT TAACATTGAC TG - #TATTAGAT        720                                                                          - ATCGTTGCTC TTTTTCCGAA CTACGATGCT AGAGCATATC CAATTCAAAC GT - #CATCCCAA        780                                                                          - TTAACAAGGG AAATTTATAC AAGTTCAGTA ATTGAAGATT CTCCAGTTTC TG - #CTAATATA        840                                                                          - CCTAATGGTT TTAATAGAGC GGAATTTGGA GTTAGACCGC CCCATCTTAT GG - #ACTTTATG        900                                                                          - AATTCTTTGT TTGTAACTGC AGAGACTGTT AGAAGTCAAA CTGTGTGGGG AG - #GACACTTA        960                                                                          - GTTAGTTCAC GAAATACGGC TGGTAACCCT ATAAATTTCC CTATTTATGG GG - #TCTTCAAT       1020                                                                          - CCTGGTGGCG CCATTTGGAT TGCAGATGAG GATCCACGTC CTTTTTATCG GA - #CATTATCA       1080                                                                          - GATCCTGTTT TTGTCCGAGG AGGATTTGGG GATCCTCATT ATGTACTTGG GC - #TTAGGGGA       1140                                                                          - GTAGGATTTC AACAAACTGG TACGAACCAC ACCCGAACAT TTAGAAATAG TG - #GGACCATA       1200                                                                          - GATTCTCTAG ATGAAATCCC ACCTCAGGAT AATAGTGGGG CACCTTGGAA TG - #ATTATAGT       1260                                                                          - CATGTATTAA ATCATGTTAC ATTTGTAAGG TGGCCTGGTG AGATTGCAGG AA - #GTGATTCA       1320                                                                          - TGGAGAGCGC CAATGTTTTC TTGGACACAC CGTAGTGCAG ATCGTACAAA TA - #TCATTAAT       1380                                                                          - CCAAATATAA TTACACAAAT ACCTGCTGTA AAAGCACACA ATCTTCATTC GG - #GTTCTACG       1440                                                                          - GTTGTTAGAG GACCCGGGTT TACAGGTGGT GATCTCTTAC GAAGAACGAA TA - #CTGGTACA       1500                                                                          - TTTGCAGATA TAAGAGTAAA TATTACTGGG CCATTATCTC AAAGATATCG TG - #TAAGAATT       1560                                                                          - CGCTATGCTT CTACGACAGA TTTACAATTT TTCACGAGAA TCAATGGAAC TT - #CTGTAAAT       1620                                                                          - CAAGGTAATT TCCAAAGAAC TATGAATAGA GGGGATAATT TAGAATCTGG AA - #ACTTTAGG       1680                                                                          - ACTGCAGGAT TTAGTACGCC TTTTAGTTTT TCAAATGCGC AAAGTACATT CA - #CATTGGGT       1740                                                                          - ACTCAGGCTT TTTCAAATCA GGAAGTTTAT ATAGATCGAA TTGAATTTGT CC - #CGGCAGAA       1800                                                                          - GTAACATTCG AGGCAGAATC TGATTTAGAA AGAGCGCAAA AGGCGGTGAA TG - #CCCTGTTT       1860                                                                          - ACTTCTACAA GCCAACTAGG GCTAAAAACA AATGTAACGG GTTACCATAT TG - #ATCAAGTG       1920                                                                          - TCCAATTTAG TTGCGTGTTT ATCGGATGAA TTTTGTCTGG ATGAAAAGAG AG - #AATTGTCC       1980                                                                          - GAGAAAGTTA AACATGCGAA GCGACTCAGT GATAAGCGGA ATTTACTTCA AG - #ATCCAAAC       2040                                                                          - TTCAGAGGGA TCAATAGGCA ACCAGACCAT GGCTGGAGAG GAAGTACGGA TA - #TTACTATC       2100                                                                          - CAAGGAGGAG ATGACGTATT CAAAGAGAAT TACGTTACGC TACCGGGTAC TT - #TTGATGAG       2160                                                                          - TGCTATCCAA CGTATTTATA TCAAAAAATA GATGAGTCGA AATTAAAAGC CT - #ATACCCGT       2220                                                                          - TATCAATTAA GAGGGTATAT CGAAGATAGT CAAGACTTAG AAATCTATTT AA - #TTCGTTAC       2280                                                                          - AATTCAAAAC ACGAAATAGT AAATGTACCA GGTACAGGGA GTTTATGGCC TC - #TTTCTGTA       2340                                                                          - GAAAATCAAA TTGGACCTTG TGGAGAACCG AATCGATGCG CGCCACACCT TG - #AATGGAAT       2400                                                                          - CCTGATTTAC ACTGTTCCTG CAGAGACGGG GAAAAATGTG TGCATCATTC TC - #ATCATTTC       2460                                                                          - TCTTTGGACA TTGATGTCGG ATGTACAGAT TTAAATGAGG ACCTAGGTGT AT - #GGTTGATA       2520                                                                          - TTCAAGATTA AGACGCAAGA TGGCCACGCA AGACTAGGGA ATCTAGAGTT TC - #TCGAAGAG       2580                                                                          - GAACCGTTAT TAGGCGAAGC GTTAGGACGT GTGAAGAGAG CGGAGAAGAA GT - #GGAGAGAC       2640                                                                          - AAACGCGAGA AACTGCAGTT GGAAACAAAT ATTGTCTATA AAGAGGCAAA AG - #AATCTGTA       2700                                                                          - GATGCTTTAT TTGTAAACTC TCAATATGAT AGATTACAAG CGGATACGAA CA - #TCGCGATG       2760                                                                          - ATTCATGCGG CAGATAAACG CGTTCATAGA ATCCGGGAAG CGTATCTGCC AG - #AGTTGTCT       2820                                                                          - GTGATTCCAG GTGTCAATGC GGCCATTTTC GAAGAATTAG AGGGACGTAT TT - #TTACAGCG       2880                                                                          - TATTCCTTAT ATGATGCGAG AAATGTTATT AAAAATGGCA ATTTCAATAA TG - #GCTTATTA       2940                                                                          - TGCTGGAACG TGAAAGGGCA TGTAGATGTA GAAGAGCAAA ACAACCACCG TT - #CGGTCCTT       3000                                                                          - GTTGTTCCGG AATGGGAAGC AGAAGTGTCA CAAGAAGTTC GTGTCTGTCC GG - #GTCGTGGC       3060                                                                          - TATATCCTTC GTGTCACAGC GTACAAAGAG GGATATGGAG AAGGCTGCGT AA - #CTATTCAT       3120                                                                          - GAAGTCGATA ATAATACAGA CGAATTGAAG TTTAGCAACT GTGAGAAAGA AC - #AAGTATAT       3180                                                                          - CCAGGTAATA CGGTAGCATG TAATGATTAT AATAAGAATC ACGGTGCGAA TG - #CATGTAGT       3240                                                                          - TCTCGTAATC GTGGATATGA CGAATCTTAT GAAAGTAATT CTTCCATACC AG - #CTGATTAT       3300                                                                          - GCACCGGTTT ATGAAGAAGA AGCGTATACA GATGGACAAA GAGGGAATCC TT - #GTGAATTT       3360                                                                          - AACAGAGGGC ATACACCATT ACCAGCTGGT TATGTGACAG CAGAGTTAGA GT - #ACTTCCCA       3420                                                                          - GAAACGGATA CAGTATGGGT TGAGATTGGA GAAACGGAAG GAACATTTAT CG - #TGGACAGT       3480                                                                          #              3504TGGA GGAA                                                  - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1168 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -    (iii) HYPOTHETICAL: YES                                                  -     (iv) ANTI-SENSE: NO                                                     -     (vi) ORIGINAL SOURCE:                                                   #THURINGIENSISORGANISM: BACILLUS                                                        (B) STRAIN: Morrissoni                                              #PS91C2   (C) INDIVIDUAL ISOLATE:                                             -    (vii) IMMEDIATE SOURCE:                                                   11 LIBRARY OF TERESA: LAMBDAGEM                                                             THOMPSON                                                                 (B) CLONE: 91C2                                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Lys Asn Asn Ile Gln Asn Gln Cys Val Pr - #o Tyr Asn Cys Leu Ser         #                15                                                           - Asn Pro Glu Val Glu Ile Leu Ser Glu Glu Ar - #g Ser Thr Gly Arg Leu         #            30                                                               - Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Ph - #e Leu Leu Ser Glu Phe         #        45                                                                   - Val Pro Gly Val Gly Val Ala Phe Gly Leu Ph - #e Asp Leu Ile Trp Gly         #    60                                                                       - Phe Ile Thr Pro Ser Glu Trp Ser Leu Phe Le - #u Leu Gln Ile Glu Gln         #80                                                                           - Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Ar - #g Asn Arg Ala Ile Thr         #                95                                                           - Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Va - #l Tyr Leu Glu Ala Leu         #           110                                                               - Arg Glu Trp Glu Glu Asn Pro Asn Asn Ala Gl - #n Leu Arg Glu Asp Val         #       125                                                                   - Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Le - #u Ile Thr Ala Ile Asn         #   140                                                                       - Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Le - #u Leu Ser Val Tyr Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Ala Ala Asn Leu His Leu Ser Leu Leu Ar - #g Asp Ala Val Ser Phe         #               175                                                           - Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Va - #l Asn Asn His Tyr Asn         #           190                                                               - Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Gl - #u His Cys Leu Asp Thr         #       205                                                                   - Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Th - #r Asn Thr Arg Gln Trp         #   220                                                                       - Ser Arg Phe Asn Gln Phe Arg Arg Glu Leu Th - #r Leu Thr Val Leu Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Val Ala Leu Phe Pro Asn Tyr Asp Ala Ar - #g Ala Tyr Pro Ile Gln         #               255                                                           - Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Th - #r Ser Ser Val Ile Glu         #           270                                                               - Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gl - #y Phe Asn Arg Ala Glu         #       285                                                                   - Phe Gly Val Arg Pro Pro His Leu Met Asp Ph - #e Met Asn Ser Leu Phe         #   300                                                                       - Val Thr Ala Glu Thr Val Arg Ser Gln Thr Va - #l Trp Gly Gly His Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Ser Ser Arg Asn Thr Ala Gly Asn Pro Il - #e Asn Phe Pro Ile Tyr         #               335                                                           - Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Il - #e Ala Asp Glu Asp Pro         #           350                                                               - Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Va - #l Phe Val Arg Gly Gly         #       365                                                                   - Phe Gly Asp Pro His Tyr Val Leu Gly Leu Ar - #g Gly Val Gly Phe Gln         #   380                                                                       - Gln Thr Gly Thr Asn His Thr Arg Thr Phe Ar - #g Asn Ser Gly Thr Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp As - #n Ser Gly Ala Pro Trp         #               415                                                           - Asn Asp Tyr Ser His Val Leu Asn His Val Th - #r Phe Val Arg Trp Pro         #           430                                                               - Gly Glu Ile Ala Gly Ser Asp Ser Trp Arg Al - #a Pro Met Phe Ser Trp         #       445                                                                   - Thr His Arg Ser Ala Asp Arg Thr Asn Ile Il - #e Asn Pro Asn Ile Ile         #   460                                                                       - Thr Gln Ile Pro Ala Val Lys Ala His Asn Le - #u His Ser Gly Ser Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Val Arg Gly Pro Gly Phe Thr Gly Gly As - #p Leu Leu Arg Arg Thr         #               495                                                           - Asn Thr Gly Thr Phe Ala Asp Ile Arg Val As - #n Ile Thr Gly Pro Leu         #           510                                                               - Ser Gln Arg Tyr Arg Val Arg Ile Arg Tyr Al - #a Ser Thr Thr Asp Leu         #       525                                                                   - Gln Phe Phe Thr Arg Ile Asn Gly Thr Ser Va - #l Asn Gln Gly Asn Phe         #   540                                                                       - Gln Arg Thr Met Asn Arg Gly Asp Asn Leu Gl - #u Ser Gly Asn Phe Arg         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Thr Ala Gly Phe Ser Thr Pro Phe Ser Phe Se - #r Asn Ala Gln Ser Thr         #               575                                                           - Phe Thr Leu Gly Thr Gln Ala Phe Ser Asn Gl - #n Glu Val Tyr Ile Asp         #           590                                                               - Arg Ile Glu Phe Val Pro Ala Glu Val Thr Ph - #e Glu Ala Glu Ser Asp         #       605                                                                   - Leu Glu Arg Ala Gln Lys Ala Val Asn Ala Le - #u Phe Thr Ser Thr Ser         #   620                                                                       - Gln Leu Gly Leu Lys Thr Asn Val Thr Gly Ty - #r His Ile Asp Gln Val         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Ser Asn Leu Val Ala Cys Leu Ser Asp Glu Ph - #e Cys Leu Asp Glu Lys         #               655                                                           - Arg Glu Leu Ser Glu Lys Val Lys His Ala Ly - #s Arg Leu Ser Asp Lys         #           670                                                               - Arg Asn Leu Leu Gln Asp Pro Asn Phe Arg Gl - #y Ile Asn Arg Gln Pro         #       685                                                                   - Asp His Gly Trp Arg Gly Ser Thr Asp Ile Th - #r Ile Gln Gly Gly Asp         #   700                                                                       - Asp Val Phe Lys Glu Asn Tyr Val Thr Leu Pr - #o Gly Thr Phe Asp Glu         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Cys Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile As - #p Glu Ser Lys Leu Lys         #               735                                                           - Ala Tyr Thr Arg Tyr Gln Leu Arg Gly Tyr Il - #e Glu Asp Ser Gln Asp         #           750                                                               - Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Ser Ly - #s His Glu Ile Val Asn         #       765                                                                   - Val Pro Gly Thr Gly Ser Leu Trp Pro Leu Se - #r Val Glu Asn Gln Ile         #   780                                                                       - Gly Pro Cys Gly Glu Pro Asn Arg Cys Ala Pr - #o His Leu Glu Trp Asn         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Pro Asp Leu His Cys Ser Cys Arg Asp Gly Gl - #u Lys Cys Val His His         #               815                                                           - Ser His His Phe Ser Leu Asp Ile Asp Val Gl - #y Cys Thr Asp Leu Asn         #           830                                                               - Glu Asp Leu Gly Val Trp Leu Ile Phe Lys Il - #e Lys Thr Gln Asp Gly         #       845                                                                   - His Ala Arg Leu Gly Asn Leu Glu Phe Leu Gl - #u Glu Glu Pro Leu Leu         #   860                                                                       - Gly Glu Ala Leu Gly Arg Val Lys Arg Ala Gl - #u Lys Lys Trp Arg Asp         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Lys Arg Glu Lys Leu Gln Leu Glu Thr Asn Il - #e Val Tyr Lys Glu Ala         #               895                                                           - Lys Glu Ser Val Asp Ala Leu Phe Val Asn Se - #r Gln Tyr Asp Arg Leu         #           910                                                               - Gln Ala Asp Thr Asn Ile Ala Met Ile His Al - #a Ala Asp Lys Arg Val         #       925                                                                   - His Arg Ile Arg Glu Ala Tyr Leu Pro Glu Le - #u Ser Val Ile Pro Gly         #   940                                                                       - Val Asn Ala Ala Ile Phe Glu Glu Leu Glu Gl - #y Arg Ile Phe Thr Ala         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Tyr Ser Leu Tyr Asp Ala Arg Asn Val Ile Ly - #s Asn Gly Asn Phe Asn         #               975                                                           - Asn Gly Leu Leu Cys Trp Asn Val Lys Gly Hi - #s Val Asp Val Glu Glu         #           990                                                               - Gln Asn Asn His Arg Ser Val Leu Val Val Pr - #o Glu Trp Glu Ala Glu         #      10050                                                                  - Val Ser Gln Glu Val Arg Val Cys Pro Gly Ar - #g Gly Tyr Ile Leu Arg         #  10205                                                                      - Val Thr Ala Tyr Lys Glu Gly Tyr Gly Glu Gl - #y Cys Val Thr Ile His         #               10401030 - #                1035                              - Glu Val Asp Asn Asn Thr Asp Glu Leu Lys Ph - #e Ser Asn Cys Glu Lys         #              10550                                                          - Glu Gln Val Tyr Pro Gly Asn Thr Val Ala Cy - #s Asn Asp Tyr Asn Lys         #          10705                                                              - Asn His Gly Ala Asn Ala Cys Ser Ser Arg As - #n Arg Gly Tyr Asp Glu         #      10850                                                                  - Ser Tyr Glu Ser Asn Ser Ser Ile Pro Ala As - #p Tyr Ala Pro Val Tyr         #  11005                                                                      - Glu Glu Glu Ala Tyr Thr Asp Gly Gln Arg Gl - #y Asn Pro Cys Glu Phe         #               11201110 - #                1115                              - Asn Arg Gly His Thr Pro Leu Pro Ala Gly Ty - #r Val Thr Ala Glu Leu         #              11350                                                          - Glu Tyr Phe Pro Glu Thr Asp Thr Val Trp Va - #l Glu Ile Gly Glu Thr         #          11505                                                              - Glu Gly Thr Phe Ile Val Asp Ser Val Glu Le - #u Leu Leu Met Glu Glu         #      11650                                                                  - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 19 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 # 19               ATA                                                        - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 36 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 #       36         TTAA TAATGCACAA TTAAGG                                     - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 25 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 #               25 ATAR TCCGT                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 23 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 #                23TTTG CGC                                                   - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Ser Thr Gly Arg Leu Pro Leu Asp                                             #5                                                                            - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #                24CRTT RGAY                                                  - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Glu Asp Ser Pro Val Ser Ala Asn                                             #5                                                                            - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 24 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                #                24CWGC WAAT                                                  - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Asn Gly Phe Asn Arg Ala Glu Phe Gly Val                                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 31 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #          31      CTGA ATTTGGGAGT W                                          - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                - Val Thr Ala Glu Thr Val Arg Ser Gln Thr                                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 30 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #           30     TWAG WAGTCAAACW                                            - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                - Val Phe Asn Pro Gly Gly Ala Ile Trp Ile Al - #a Asp Glu                     #10                                                                           - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 42 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                #  42              GMGC MATWTGGATW GCWGATGARG AT                              - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                - Val Arg Gly Gly Phe Gly                                                     #5                                                                            - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #  18              GR                                                         - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                - Gly Thr Asn His Thr Arg Thr                                                 #5                                                                            - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #21                GAAC W                                                     - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 7 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - Val Arg Trp Pro Gly Glu Ile                                                 #5                                                                            - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 21 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #21                ARAT W                                                     - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 6 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                - Ser Asp Ser Trp Arg Ala                                                     #5                                                                            - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                                    (A) LENGTH: 18 bases                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: DNA (synthetic)                                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #  18              CW                                                         - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 1174 amino                                                        (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - Met Xaa Asn Asn Ile Gln Asn Gln Cys Val Pr - #o Tyr Asn Cys Leu Xaa         #                15                                                           - Asn Pro Glu Val Glu Ile Leu Xaa Glu Glu Ar - #g Ser Thr Gly Arg Leu         #            30                                                               - Pro Leu Asp Ile Ser Leu Ser Leu Thr Arg Ph - #e Leu Leu Ser Glu Phe         #        45                                                                   - Val Pro Gly Val Gly Val Ala Phe Gly Leu Ph - #e Asp Leu Ile Trp Gly         #    60                                                                       - Phe Ile Thr Pro Ser Xaa Trp Ser Leu Phe Le - #u Leu Gln Ile Glu Gln         #80                                                                           - Leu Ile Glu Gln Arg Ile Glu Thr Leu Glu Ar - #g Asn Arg Ala Ile Thr         #                95                                                           - Thr Leu Arg Gly Leu Ala Asp Ser Tyr Glu Xa - #a Tyr Xaa Glu Ala Leu         #           110                                                               - Arg Glu Trp Glu Xaa Asn Pro Asn Asn Ala Gl - #n Leu Arg Glu Asp Val         #       125                                                                   - Arg Ile Arg Phe Ala Asn Thr Asp Asp Ala Le - #u Ile Thr Ala Ile Asn         #   140                                                                       - Asn Phe Thr Leu Thr Ser Phe Glu Ile Pro Le - #u Leu Ser Val Tyr Val         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Gln Ala Ala Asn Leu His Leu Ser Leu Leu Ar - #g Asp Ala Val Ser Phe         #               175                                                           - Gly Gln Gly Trp Gly Leu Asp Ile Ala Thr Va - #l Asn Asn His Tyr Asn         #           190                                                               - Arg Leu Ile Asn Leu Ile His Arg Tyr Thr Xa - #a His Cys Leu Asp Thr         #       205                                                                   - Tyr Asn Gln Gly Leu Glu Asn Leu Arg Gly Th - #r Asn Thr Arg Gln Trp         #   220                                                                       - Xaa Arg Phe Asn Gln Phe Arg Arg Xaa Leu Th - #r Leu Thr Val Leu Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Ile Val Ala Leu Phe Pro Asn Tyr Asp Xaa Ar - #g Xaa Tyr Pro Ile Gln         #               255                                                           - Thr Ser Ser Gln Leu Thr Arg Glu Ile Tyr Th - #r Ser Ser Val Ile Glu         #           270                                                               - Asp Ser Pro Val Ser Ala Asn Ile Pro Asn Gl - #y Phe Asn Arg Ala Glu         #       285                                                                   - Phe Gly Val Arg Pro Pro His Leu Met Asp Ph - #e Met Asn Ser Leu Phe         #   300                                                                       - Val Thr Ala Glu Thr Val Arg Ser Gln Thr Va - #l Trp Gly Gly His Leu         305                 3 - #10                 3 - #15                 3 -       #20                                                                           - Val Ser Ser Arg Asn Thr Ala Gly Asn Xaa Il - #e Asn Phe Pro Xaa Tyr         #               335                                                           - Gly Val Phe Asn Pro Gly Gly Ala Ile Trp Il - #e Ala Asp Glu Asp Pro         #           350                                                               - Arg Pro Phe Tyr Arg Thr Leu Ser Asp Pro Va - #l Phe Val Arg Gly Gly         #       365                                                                   - Phe Gly Xaa Pro His Tyr Val Leu Gly Leu Ar - #g Gly Val Xaa Phe Gln         #   380                                                                       - Gln Thr Gly Thr Asn His Thr Arg Thr Phe Ar - #g Asn Ser Gly Thr Ile         385                 3 - #90                 3 - #95                 4 -       #00                                                                           - Asp Ser Leu Asp Glu Ile Pro Pro Gln Asp As - #n Ser Gly Ala Pro Trp         #               415                                                           - Asn Asp Tyr Ser His Val Leu Asn His Val Th - #r Phe Val Arg Trp Pro         #           430                                                               - Gly Glu Ile Xaa Gly Ser Asp Ser Trp Arg Al - #a Pro Met Phe Ser Trp         #       445                                                                   - Thr His Arg Ser Ala Xaa Xaa Thr Asn Xaa Il - #e Xaa Pro Xaa Xaa Ile         #   460                                                                       - Thr Gln Ile Pro Xaa Val Xaa Ala His Xaa Le - #u Xaa Ser Gly Xaa Thr         465                 4 - #70                 4 - #75                 4 -       #80                                                                           - Val Val Arg Gly Pro Gly Phe Thr Gly Gly As - #p Xaa Leu Arg Arg Thr         #               495                                                           - Xaa Xaa Gly Xaa Phe Ala Xaa Xaa Xaa Val As - #n Ile Xaa Gly Xaa Leu         #           510                                                               - Xaa Gln Arg Tyr Arg Xaa Arg Ile Arg Tyr Al - #a Ser Thr Thr Xaa Leu         #       525                                                                   - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xa - #a Xaa Xaa Gly Xaa Phe         #   540                                                                       - Xaa Xaa Thr Met Xaa Xaa Gly Asp Xaa Leu Xa - #a Xaa Xaa Xaa Phe Xaa         545                 5 - #50                 5 - #55                 5 -       #60                                                                           - Xaa Ala Xaa Xaa Xaa Thr Xaa Phe Xaa Phe Xa - #a Xaa Xaa Gln Ser Xaa         #               575                                                           - Phe Thr Xaa Gly Xaa Xaa Xaa Phe Xaa Ser Xa - #a Xaa Glu Val Tyr Ile         #           590                                                               - Asp Xaa Xaa Glu Xaa Xaa Pro Xaa Xaa Xaa Th - #r Phe Glu Ala Glu Xaa         #       605                                                                   - Asp Xaa Glu Arg Ala Gln Xaa Ala Val Asn Al - #a Leu Phe Thr Ser Xaa         #   620                                                                       - Xaa Gln Xaa Gly Xaa Xaa Thr Xaa Val Thr Xa - #a Tyr His Ile Asp Gln         625                 6 - #30                 6 - #35                 6 -       #40                                                                           - Val Ser Asn Leu Val Xaa Cys Leu Ser Asp Gl - #u Phe Cys Leu Asp Glu         #               655                                                           - Xaa Arg Glu Leu Ser Glu Xaa Val His Xaa Al - #a Xaa Arg Leu Ser Asp         #           670                                                               - Xaa Arg Asn Leu Leu Gln Asp Pro Asn Phe Xa - #a Gly Ile Asn Arg Gln         #       685                                                                   - Xaa Asp Xaa Gly Trp Arg Gly Ser Thr Asp Il - #e Thr Ile Gln Xaa Gly         #   700                                                                       - Asp Asp Val Phe Xaa Glu Asn Tyr Val Thr Le - #u Pro Gly Thr Phe Asp         705                 7 - #10                 7 - #15                 7 -       #20                                                                           - Glu Cys Tyr Pro Thr Tyr Leu Tyr Gln Xaa Il - #e Asp Glu Ser Xaa Leu         #               735                                                           - Xaa Xaa Tyr Thr Arg Tyr Gln Leu Arg Gly Ty - #r Ile Glu Asp Ser Gln         #           750                                                               - Asp Leu Glu Ile Tyr Leu Ile Arg Tyr Asn Xa - #a Xaa His Glu Pro Val         #       765                                                                   - Asn Val Xaa Gly Thr Gly Ser Leu Trp Pro Le - #u Ser Val Xaa Xaa Xaa         #   780                                                                       - Ile Xaa Xaa Cys Gly Glu Pro Asn Arg Cys Al - #a Pro His Leu Glu Trp         785                 7 - #90                 7 - #95                 8 -       #00                                                                           - Asn Pro Asp Leu Xaa Cys Ser Cys Arg Asp Gl - #y Glu Xaa Cys Xaa His         #               815                                                           - His Ser His His Phe Ser Leu Asp Ile Asp Va - #l Gly Cys Thr Asp Leu         #           830                                                               - Asn Glu Asp Leu Xaa Val Trp Xaa Ile Phe Xa - #a Ile Xaa Thr Gln Asp         #       845                                                                   - Gly His Ala Arg Leu Gly Asn Leu Glu Phe Le - #u Glu Glu Xaa Pro Leu         #   860                                                                       - Xaa Gly Glu Ala Leu Xaa Arg Val Xaa Arg Al - #a Glu Xaa Xaa Trp Arg         865                 8 - #70                 8 - #75                 8 -       #80                                                                           - Asp Xaa Arg Glu Xaa Leu Xaa Leu Glu Thr As - #n Ile Val Tyr Xaa Glu         #               895                                                           - Ala Xaa Glu Ser Val Asp Ala Leu Phe Val As - #n Ser Gln Tyr Asp Xaa         #           910                                                               - Leu Gln Ala Asp Thr Asn Ile Ala Met Ile Hi - #s Ala Ala Asp Xaa Arg         #       925                                                                   - Val His Arg Ile Arg Glu Ala Tyr Leu Pro Gl - #u Leu Ser Val Ile Pro         #   940                                                                       - Gly Val Asn Xaa Xaa Ile Phe Glu Glu Leu Xa - #a Gly Arg Ile Phe Thr         945                 9 - #50                 9 - #55                 9 -       #60                                                                           - Ala Xaa Xaa Leu Tyr Asp Ala Arg Asn Val Il - #e Xaa Asn Gly Xaa Phe         #               975                                                           - Asn Asn Gly Leu Xaa Cys Trp Asn Val Xaa Gl - #y His Val Asp Val Glu         #           990                                                               - Glu Gln Asn Asn His Arg Ser Val Leu Val Va - #l Pro Glu Trp Glu Ala         #      10050                                                                  - Glu Val Ser Gln Glu Val Arg Val Cys Pro Gl - #y Arg Gly Tyr Ile Leu         #  10205                                                                      - Arg Val Thr Ala Tyr Xaa Glu Gly Tyr Gly Gl - #u Gly Cys Val Thr Ile         #               10401030 - #                1035                              - His Glu Xaa Xaa Asn Asn Thr Asp Glu Leu Xa - #a Phe Ser Asn Cys Xaa         #              10550                                                          - Xaa Glu Xaa Val Tyr Pro Xaa Asn Thr Val Xa - #a Cys Asn Asp Tyr Xaa         #          10705                                                              - Xaa Asn Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xa - #a Ser Arg Asn Arg Gly         #      10850                                                                  - Tyr Asp Glu Xaa Tyr Xaa Ser Asn Ser Ser Xa - #a Pro Ala Asp Tyr Ala         #  11005                                                                      - Xaa Val Tyr Glu Glu Xaa Xaa Tyr Thr Asp Gl - #y Xaa Arg Xaa Asn Pro         #               11201110 - #                1115                              - Cys Glu Xaa Asn Arg Gly Xaa Xaa Xaa Xaa Th - #r Pro Leu Pro Ala Gly         #              11350                                                          - Tyr Val Thr Xaa Glu Leu Glu Tyr Phe Pro Gl - #u Thr Asp Xaa Val Trp         #          11505                                                              - Xaa Glu Ile Gly Glu Thr Glu Gly Thr Phe Il - #e Val Asp Ser Val Glu         #      11650                                                                  - Leu Leu Leu Met Glu Glu                                                         1170                                                                      __________________________________________________________________________

We claim:
 1. A composition of matter comprising Bacillus thuringiensisPS91C2, or a mutant thereof, or spores or crystals of said isolate, inassociation with an insecticide carrier.
 2. A substantially pure toxinprotein wherein said toxin has activity against a lepidopteran pest, andwherein said toxin comprises all or a lepidopteran-active part of SEQ IDNO.
 4. 3. The toxin of claim 2 wherein said toxin has the amino acidsequence shown in SEQ ID NO.
 4. 4. A toxin, free from naturallyassociated impurities, encoded by a nucleotide sequence obtainable fromBacillus thuringiensis PS91C2, or a portion of said nucleotide sequence,wherein said toxin is active against lepidopteran pests.
 5. A toxinactive against lepidopteran pests, wherein said toxin is free fromnaturally associated impurities, and wherein said toxin comprises anamino acid sequence shown in SEQ ID NO. 4 or a lepidopteran-activeportion thereof.